Hepatitis C virus (HCV) is a major cause of chronic liver disease, liver cirrhosis and hepatocellular carcinoma. Preliminary studies with a water-soluble, cell-permeable and non-cytotoxic synthetic peptide called DAPL (Dmt-D-Arg-Phe-Lys-NH2, where Dmt is 2', 6'- dimethyltyrosine) demonstrate that this peptide can selectively interact with the HCV RNA translation initiation site (HCV IRES) and inhibit HCV translation selectively in vitro. The goal of this proposal is to explore the biochemistry, pharmacology and mechanism of action of DAPL in inhibiting HCV translation, the key element of HCV infection. Three specific aims were established: (1) Studies on regulation of HCV-IRES-mediated translation by DAPL in vitro. Thus, we plan to demonstrate an effect of DAPL on HCV translation in vitro and determine the site(s) of DAPL-IRES interaction, by determining the sites of the IRES protected by DAPL from enzymatic, chemical, and physical probing. We will also determine the effect of DAPL on ribosomal assembly on HCV IRES, by determining the effect of DAPL on HCV RNA binding to ribosomal subunits and the ribosomal protection of HCV IRES from RNase digestion. (2) To examine the ability of DAPL to regulate HCV translation and replication in cells. Thus, the effect of DAPL on HCV IRES-directed translation and HCV RNA synthesis in Huh 7 and Huh-7.5 cells transfected with DNA vectors carrying reporters (luciferase) and HCV replicon, respectively will be determined with Northern and Western assays. (3) To investigate structural requirements of DAPL for selective inhibition of HCV translation. Thus, we plan to investigate the role of dimethyl-tyrosine, arginine, and different lengths terminal lysines in the regulation of HCV translation in vitro and their interaction with HCV IRES. If DAPL or related analogues can be further developed to target and inhibit HCV RNA translation, the clinical application of this kind of therapeutics might be significant.